The present invention relates to compositions and methods to deliver therapeutic agents to a subject in need thereof using polypeptide carriers. More particularly, the present invention relates to polypeptides such as Giardia sp. variable surface proteins (VSPs) that act as carriers for the delivery of therapeutic agents such as bioactive peptides.
Oral delivery represents the ideal means of delivering prophylactic and therapeutic agents because of ease of administration, patient compliance, and cost. However, the oral route is also the most difficult because of the numerous barriers posed by the gastrointestinal tract (GIT). The main challenges are enzymatic degradation in the stomach and upper intestinal tract, and lack of sufficient permeability through the GIT. Low pH in the stomach can subject therapeutic agents to physical and chemical degradation. Physical degradation of peptides generally involves modification of the native structure of a protein to a higher-order structure which may be a result of adsorption, aggregation, unfolding, precipitation, and/or complete/partial degradation to its amino acidic components. Chemical degradation usually involves blond cleavage and leads to the formation of a new product.
Giardia is an intestinal pathogen which is capable of surviving the harsh environmental conditions in the stomach and the upper small intestine. Like many protozoan microorganisms, Giardia undergoes antigenic variation (see, e.g., Zambrano-Villa et al., Trends Parasitol. 18: 272-8 (2002)), a mechanism by which it continuously switches its major surface molecules allowing the parasite to evade the host's immune response and establish chronic and/or recurrent infections (see, e.g., Nash, Mol. Microbiol. 45:585-90 (2002)). These surface antigens belong to a family of Variant-specific Surface Proteins (VSPs), which are integral membrane proteins that cover the entire surface of trophozoites.
VSPs possess a cysteine-rich amino-terminal region, and a conserved carboxy-terminal domain including a transmembrane region and a short cytoplasmic tail (FIG. 1C). There is a repertoire of about 200 VSP genes in the Giardia's genome, but only one VSP is expressed on the surface of the parasite at any given time (see, e.g., Prucca et al., Nature 456(7223):750-4 (2008); Deitsch et al., Microbiol. Mol. Biol. Rev. 61:281-93 (1997)).
Since the extracellular portion of Giardia VSPs allows the parasite to survive within the hostile environment of the upper small intestine, VSPs covalently bound to antigens have been used to shuttle candidate antigens. It has been observed that when vaccines comprising VSPs covalently bound to Giardia antigens are administered orally, the vaccines fully protect animals from subsequent infections by the Giardia parasite, indicating that the antigens have survived the passage through the GIT (Rivero et al., Nat. Med. 16(5):551-7 (2010), see, e.g., PCT Pub. Nos. WP2010/064204 and WO2011/120994, which are herein incorporated by reference in their entireties).
Type 1 diabetes is usually diagnosed in children and young adults, and is responsible for a growing proportion of national health care expenditures. Type 1 diabetes is an autoimmune disease that results from a dysfunction of the immune system that attacks and destroys the β-cells of the pancreatic islets producing insulin.
Administration of exogenous insulin is the only medication that can be used to control the increases in blood sugar that occur with the disease. Type 2 diabetes, by contrast, is characterized by defects in both insulin secretion and insulin action, with insulin deficiency usually emerging later during the course of the disease. Insulin supplementation is often required to attain good glucose levels control in this disease (see, e.g., DeWitt & Hirsch, JAMA 289:2254-2264 (2003)). There are different types of subcutaneous insulin available (Summers et al., Clin. Ther. 26:1498-1505 (2004)). However, surveys indicate substantial resistance to insulin therapy on the part of patients with type 2 diabetes due to anticipated pain and inconvenience (Peyrot et al., Diabetes Care 28:2673-2679 (2005)). The youngest and oldest patients are least likely to accept injectable therapy and thus pose the greatest challenge for physicians who want to initiate insulin treatment (Freemantle et al., Diabetes Care 28:427-428 (2005)). Consequently, efforts to develop oral, nasal, and inhaled formulations of insulin have been driven by the preference of patients to avoid subcutaneous injections (Cefalu, Ann. Med. 33:579-586 (2001); Graham et al., N. Engl. J. Med. 356:497-502 (2007)). Thus, the option of delivering insulin by the oral route remains an attractive therapeutic strategy.
Glucagon, a peptide hormone secreted by the pancreas, raises blood glucose levels. Its effect is opposite that of insulin, which lowers blood glucose levels. Glucagon is indicated and used as a treatment for severe hypoglycemia. Because patients with type 1 diabetes may have less of an increase in blood glucose levels compared with a stable type 2 patient, supplementary carbohydrates should be given as soon as possible, especially to a pediatric patient. Glucagon is also indicated as a diagnostic aid in the radiologic examination of the stomach, duodenum, small bowel, and colon when diminished intestinal motility would be advantageous. Glucagon is as effective for this examination as are the anticholinergic drugs. However, the addition of the anticholinergic agent may result in increased side effects. As in the case of insulin, the development of forms of glucagon suitable for oral delivery is an attractive therapeutic strategy.
Growth hormone deficiency is a disorder that involves the pituitary gland, which produces growth hormone and other hormones. Human growth hormone (hGH) stimulates growth and cell reproduction in humans, also exerting its action on metabolism of lipids, proteins and carbohydrates. Recombinant hGH is commonly produced by bacterial fermentation (Zeisel et al., Horm. Res. 37(Suppl 2):5-13 (1992); Sonoda & Sigimura, Biosci. Biotechnol. Biochem. 72:2675-80 (2008)). When the pituitary gland does not produce enough growth hormone, growth will be slower than normal. Growth hormone is needed for normal growth in children. In adults, growth hormone is needed to maintain the proper amounts of body fat, muscle, and bone. hGH deficiency can occur at any age. Children and some adults with growth hormone deficiency will benefit from growth hormone therapy. To treat growth hormone deficiencies, hGH (human growth hormone) is generally prescribed. hGH is an injectable drug which is injected underneath the fat of the patient's skin several times a week (Brearley et al., BMC Clin. Pharmacol. 7:10 (2007)). As in the case of insulin treatment, patient's resistance to the initiation injectable therapy and compliance pose challenges for physicians. Accordingly, the development of hGH forms suitable for oral delivery is an attractive therapeutic strategy.